Int.J.Curr.Microbiol.App.Sci (2015) 4(12): 795-802
ISSN: 2319-7706 Volume 4 Number 12 (2015) pp. 795-802
http://www.ijcmas.com
Original Research Article
Genotyping exon 1 and Partial Intron 1 Region of Caprine KISS1 Gene
using Acl I, Sac II and Dra III
Rohita Gupta*, A.P Singh, A Prajapati, J Namdev and B.C Sarkhel
Animal Biotechnology Center, Nanaji Deshmukh Veterinary Science University, Jabalpur, India
*Corresponding author
ABSTRACT
Keywords
KISS 1,
Polymorphism,
Black Bengal
goat,
PCR-RFLP,
Genotyping
In a view of the importance of KISS1 as a regulator of puberty onset.
Polymorphisms of KISS1 was studied to understand the relationship with high
prolificacy and sexual precocity of Black Bengal goat breed. The various genotypes
of KISS1 gene were identified using RFLP and gene and genotype frequencies
were estimated. The maximum gene frequency for Black Bengal goat was observed
for G allele (93%) of Sac II RE where as it was minimum for T allele (3%) at the
same locus. The gene frequency of G allele was 80% and for C allele was 20% for
Acl I RE. The gene frequency of T allele was 39% and for A allele was 61% for
Dra III RE. The maximum genotype frequency was found in GG genotype (93%)
of Black Bengal goat by Sac II RE where as it was minimum for GT allele (7%) at
the same locus. The genotype frequency of GG was 60% and for GC was 40% for
Acl I RE. The genotype frequency of TT, TA and AA was 14%, 50% and 36%
respectively for Dra III RE. In present investigation it was found that frequency of
allele G and T was high and favoured by the natural selection. It can be concluded
that three variations in KISS1 gene may play an important role in litter size in
native goat breeds of Black Bengal but further research on a large population will
be required to confirm the linkage with increased prolificacy in goats.
Introduction
(An et al. 2013), INHA and INHBA (Hou et
al. 2012), KISS1 (Cao et al. 2010, Cao et al.
2011 and An et al. 2012), KITLG (An et al.
2013), POUF1 (Feng et al. 2012) and TSHB
(Huang et al. 2013) genes, amongst others,
has been screened and exciting opportunity
to add a high level of prolificacy to sheep
and goat.
The Black Bengal goat is a highly prolific
and major meat producing animal in West
Bengal along with the adjoining part of the
Jharkhand, Orissa, Bihar, Tripura states of
India (Zeshmarani et al. 2007). Twins and
triplets are common in kidding of Black
Bengal goat. An investigation into genetic
factors responsible for their high prolificacy,
fertility, early sexual maturity has not been
much studied. The fecundity genes such as
(Chu et al. 2011), CART (Wang et al.
2011), GDF9 (Feng et al. 2011), GNRH1
These genes have proved to be traceable and
persistent (Abraham and Thomas, 2012).
The KISS1 gene encodes a family of
795
Int.J.Curr.Microbiol.App.Sci (2015) 4(12): 795-802
neuropeptides called kisspeptins, which
activate
receptor
G
protein-coupled
receptor-54 and play a role in the
neuroendocrine regulation of GnRH
secretion (Smith et al. 2006). Kisspeptins
are very potent elicitors of luteinizing
hormone (LH) and follicle-stimulating
hormone (FSH) secretion in different
mammalian species (Gottsch et al. 2004;
Dhillo et al. 2005). Specifically in the
female, the hypothalamic KISS1/ GPR54
system seems to operate as a central conduit
for not only the negative but also the
positive
feedback
regulation
of
gonadotropins, thereby playing a substantial
role in the generation of the preovulatory
surge of LH (An et al. 2013) These findings
suggest that the KISS1 gene could be an
excellent candidate gene for reproductive
traits in caprine (Ohtaki et al. 2001). Genetic
improvement of polygenic traits can be
enhanced by marker assisted selection which
has higher accuracy in estimating the genetic
value of animals (Dekker, 2004).
(A260/A280 ratio, i.e absorbance at
wavelengths of 260 and 280) of DNA was
assessed by using a NanoDrop 2000
Spectrophotometer (Thermo Scientific,
Waltham, MA) and gel electrophoresis. The
genomic DNA concentration ranged from
270-2278 ng/µl and diluted upto 50 ng/µl
with nuclease free water and stored at -200 C
for further use.
Polymerase Chain Reaction (PCR) for
KISS 1 Gene
In the present study the exon 1 and partial
intron 1 region of 1101 bp of KISS1 gene
was selected for SNP investigation and
genotyping. On the basis of the caprine
KISS1 gene sequence (GenBank accession
No. GU142847), pairs of primers (F: 5 CCT GTG TTT GCT GGA CAG TCT and
R: 5 - TGC TCC CTC CCA ACC TTC TT)
was selected to amplify the exon 1 and
partial intron 1 of the KISS1 gene (Cao et al.
2010). The 25- L reaction volume contained
50 ng genomic DNA, 12.5 L 2X reaction
mix (Fermentas) and 0.5 M of each primer.
The cycling protocol was 5 min at 95°C and
35 cycles of denaturation at 94°C for 30 s,
annealing at 64 °C for 30 s, and extension at
72°C for 35 s, with a final extension at 72°C
for 10 min. PCR amplification was
confirmed by running, 10 µl of PCR product
mixed with 6X gel loading dye from each
tube on 1.5 % agarose gel at a constant
voltage 80 V for 50 to 60 min in 0.5X TBE
buffer. The amplified product was visualized
as a single compact band of expected size
under UV light and documented by gel
documentation system (Syngene, Gene
Genius Bio Imaging).
Materials and Methods
Genomic DNA Isolation
Five ml of blood sample was collected from
total eighteen Black Bengal goats
maintained at goat breeding farm, NDVSU,
Jabalpur, where all diets were uniform and
health, fertility and production records were
maintained. Blood was collected from
jugular vein, using EDTA coated vacutainer
tube separately for each sample. The
collected blood samples were brought to the
laboratory on ice. The genomic DNA was
extracted from white blood cells using a
standard
phenol-chloroform
extraction
protocol (Sambrook et al, 1989). All experiments were performed in accordance with
the animal ethics committee of the
institution where the experiment was
conducted. The quantity and quality
Restriction
Products
Digestion
of
the
PCR
The SNPs of the KISS1 gene were
genotyped by polymerase chain reaction796
Int.J.Curr.Microbiol.App.Sci (2015) 4(12): 795-802
restriction fragment length polymorphism
(PCR-RFLP) using the restriction enzymes
Acl I, Sac II and Dra III
(Thermo
scientific). The PCR products 10µl from
each tube were digested with 1 U restriction
enzymes (Acl I /Sac II /Dra III), in the
manufacturer s 10 X assay buffer in the final
reaction volume of 30µl. The reaction
mixture was centrifuged for few seconds for
uniform mixing and then incubated at 37 C
for 4 hours in the thermo Cycler and
deactivation at 65 C at 20 min.
Genotype frequency =
Total number of individuals of particular
genotypes
-----------------------------------------------------Total number of individuals of all genotypes
Results and Discussion
PCR amplification of Exon 1 and partial and
intron 1 region of KISS1 gene was used as
target to reveal the polymorphism. The PCR
amplification was carried out and the
amplified product of KISS 1 gene was
subjected to electrophoreses on 1.5%
agarose gel and a single clear band of 1101
bp was obtained (Fig. 1).
Analysis of the PCR-RFLP Products
After restriction digestion, the PCR products
were electrophoreses on 1.5 % agarose gel
(according to the expected size of
fragments) adding 5 µl ethidium bromide in
100ml solution. A constant voltage of 80V
for 60 minutes was applied to gel
electrophoresis apparatus, using 0.5X TBE
buffer. The digested PCR samples, 6X
loading dye and GeneRuler
(Fermentas
life sciences) 100bp DNA Ladder (Range,
80-1013 bp) was used as a molecular size
marker. The bands were visualized under
UV light and documented by gel
documentation system. The RFLP product
were determined by comparing with
molecular size marker and the genotypes
were then determined.
Three restriction enzymes used for
restriction digestion of KISS 1 gene (1101
bp) were Acl I, Sac II and Dra III. The
amplified PCR product was digested with
Acl1 enzyme and revealed GG and GC
genotypes in Black Bengal goat breed. The
band size for GG genotype was 1101 bp and
for GC were 1101, 805 and 296 bp.
Restriction enzyme, Sac II revealed two
genotypes, GG and GT genotype.
Homozygous genotype GG revealed a band
size of 1101 bp whereas heterozygous
genotype GT revealed a band size of 1101,
647 and 454 bp. After the digestion with the
last enzyme, Dra III, the three genotypes
TT, TA and AA were obtained.
Homozygous genotype TT reveals a band
size of 1101 bp whereas heterozygous
genotype TA revealed a band size of 1101,
598 and 503 bp and homozygous AA
revealed a band size of 598 and 503 bp
(Fig.2)
Gene and Genotype Frequency
The gene and genotype frequencies were
detected by studying the presence or absence
of specific size band patterns. Before
genotyping these band patterns were
compared with available literature and in
addition molecular size of the bands was
confirmed by comparing with 100 bp
molecular marker.
The frequencies
calculated.
of
genotypes
Gene and Genotype Frequency of KISS1
Gene
were
Gene frequencies and genotype frequencies
for KISS1 gene have been summarized in
797
Int.J.Curr.Microbiol.App.Sci (2015) 4(12): 795-802
Table 1 and 2 respectively. The maximum
gene frequency for Black Bengal goat was
observed for G allele (93%) of Sac II RE
where as it was minimum for T allele (3%)
at the same locus. The gene frequency of G
allele was 80% and for C allele was 20% for
Acl I RE. The gene frequency of T allele
was 39% and for A allele was 69% for Dra
III RE.
frequency of GG genotype (49%). However
Cao et al., (2010) also reported that the
frequency of GT genotype was higher than
GG genotype in JG and IMC goat breeds
and TT genotype was higher in BG and
WDG goat breeds.
The 1101 bp amplified product after
digestion with Dra III revealed three
genotypes. The frequency of TA genotype
was higher than AA and TT genotype. The
allelic frequency of A allele was higher than
T allele. Similar finding was reported by
Cao et al. (2010) that TA genotype has high
genotype frequency in LC and IMC goat
breeds.
The maximum genotype frequency was
found in GG genotype (93%) of Black
Bengal goat by Sac II RE where as it was
minimum for GT allele (6.7%) at the same
locus. The genotype frequency of GG was
60% and for GC was 40% for Acl I RE. The
genotype frequency of TT, TA and AA was
14%, 50% and 36% respectively for Dra III
RE.
The lower frequency of GC, GT genotype
and absence of CC, TT genotype may be
due to unfavorable natural selection. This
may also be due to small population size
taken under the present investigation. In
present investigation it was found that
frequency of allele G and T was high and
favoured by the natural selection.
PCR-RFLP analysis of KISS1 gene after
digestion with Acl I revealed two genotypes
GG and GC in Black Bengal breed of goat.
However homozygous CC genotype was not
seen in these breeds of goat. The frequency
of GG genotype was higher than GC
genotype. The allelic frequency of G allele
was higher than C allele. Similar findings
were also reported by Cao et al. (2010)
showing higher frequency of GG genotype
in JG (Jining Grey) breed (82%), LC
(Liaoning Cashmere) breed (77%) and IMC
(Inner Mongolia Cashmere), BG (Boer) and
WDG (Wendeng dairy ) breed (83%) of
goats.
Identification of the candidate genes which
are responsible for variation in quantitative
traits has been a challenge in modern
genetics. So far, there are few reports of
KISS1 as a candidate gene for reproductive
traits in animals, which revealed that KISS1
gene plays an important role in animal
reproduction Tomikawa et al. (2010).
In the present investigation three
polymorphism were detected in KISS1 gene
for Black Bengal goat breed by PCR-RFLP
using three different REs. An et al. (2013a)
reported 10 polymorphism in three Chinese
goat breeds SN, GZ and BG g.1147T>C,
g.1417G>A, g.1428_1429delG, g.2124C>T,
g.2270C>T,
g.2489T>C,
g.2510G>A,
g.2540C>T, g.3864_3 865delCA and
g.3885_ 3886 ins ACCCC.
The 1101 bp amplified product after
digestion with Sac II restriction enzyme
revealed two genotypes i.e., GG, GT in
Black Bengal breed of goat. However
homozygous TT genotype was absent. The
frequency of GG genotype was higher than
GT genotype. The allelic frequency of G
allele was higher than allelic frequency of
T . Similar findings in LC goat breed was
reported by Cao et al. (2010) showing high
798
Int.J.Curr.Microbiol.App.Sci (2015) 4(12): 795-802
Table.1 Gene frequency of KISS I gene of black Bengal breed
RE
Acl I
Allele
G
C
Gene frequency
0.8
0.2
Percentage
80%
20%
Sac II
G
T ..
0.97
0.03
97%
3%
Dra III
T
A
0.39
0.61
39%
61%
Table.2 Genotype frequency of KISS I gene of black Bengal breed
RE
Acl I
296bp
Genotype
G G(9)
G C(6)
Genotype Frequency
0.6
0.4
Percentage
60%
40%
Sac II
454bp
GG(14)
GT(1)
0.933
0.067
93.3%
6.7%
Dra III
504bp
TT(2)
TA(7)
AA(5)
0.14
0.5
0.36
14%
50%
36%
Fig.1 PCR amplified product of KISS 1 gene of 1101 bp in Agarose gel electrophoresis (1.5 %)
799
Int.J.Curr.Microbiol.App.Sci (2015) 4(12): 795-802
Fig.2 RE digestion ( Acl I, Sac II and Dra III ) of KISS 1 gene of exon 1 and partial Intron 1
(1101bp) in agarose gel electrophoresis (1.5 %)
L1 and L4: GCgenotype; L2 and L5: GT genotype; L3: TA genotype; L6: AA genotype
L7: 100bp DNA Ladder
An et al. (2013b) also reported two novels
SNP in three Chinese goat breeds i.e.,
Xinong Saanen (SN), Guanzhong (GZ) and
Boer (BG) at g.2124 and g.2270. Huijbregts
et al. (2012) detected three SNP (c.638 insT,
c.641C>G and c.645G>CA) in the 5 UTR of
human KISS1 gene and found the c.645
G>C mutation was associated with central
precocious puberty. Cao et al. (2010)
reported an association between allele C of
the 296 locus and allele deletion of the
1960_1977 locus of the KISS1 gene and
larger litter size in Jining goat. Hou et al.
(2011) identified T2643C and 8_bp base
deletions (2677 AGTTCCCC) in intron 2 of
the goat KISS1 gene with 82634C showing
significant affect on litter size (p 0.05).
allele G and T was high and favoured by the
natural selection. From present study it was
concluded that three variations in Kiss1 gene
may play an important role in litter size in
native goat breeds of Black Bengal. Further
research on a large size of goat population
will be required to confirm the linkage with
increased prolificacy in Black Bengal goats.
Acknowledgement
Thanks are due to the Director, Animal
biotechnology
centre,
for
providing
necessary laboratory facilities and for the
permission to collect blood sample from
goat farm.
References
In conclusion, the maximum genotype
frequency was found in GG genotype (93%)
of Black Bengal goat by Sac II RE where as
it was minimum for GT allele (6.7%) at the
same locus. The genotype frequency of TT,
TA and AA was 14%, 50% and 36%
respectively for Dra III RE. In present
investigation it was found that frequency of
Abraham A, Thomas N (2012) Role of
fecundity genes in prolificacy of
small ruminants. J Ind Vet Asso 10:
34-37.
An XP, Hou JX, Li G et al. (2012).
Polymorphism identification in the
800
Int.J.Curr.Microbiol.App.Sci (2015) 4(12): 795-802
goat KITLG gene and association
analysis with litter size. Ani Genet
(43)104 107.
An XP, Ma T, Hou JX, Fang F, Han P, Yan
Y, Zhao HB, Wang JG, Cao BY
(2013) Association analysis between
variants in KISS1 gene and litter size
in goat. BMC Genet 14:1 6.
An XP, Han P, Hou J X, Zhao H B, Yan Y
et al (2013b) Molecular cloning and
characterization of KISS1 promoter
and effect of KISS1 gene mutations
on litter size in the goat. Genet Mol
Res 12 (4): 4308-4316.
An XP, Hou JX, Zhao HB et al (2013)
Polymorphism identification in goat
GNRH1 and GDF9 genes and their
association analysis with litter size.
Ani Gen (44): 234 238.
Cao GL, Chu MX, Fang L, Di R, Feng T, Li
N (2010) Analysis on DNA sequence
of KISS-1 gene and its association
with litter size in goats. Mol Bio Rep
37 (8):3921-9.
Cao GL, Chu MX, Fang L, Feng T, Di R, Li
N (2011)Analysis on DNA sequence
of GPR54 gene and it sassociation
with litter size in goats. Mol Biol
Rep (38):3839 3848.
Chu MX, Lu L, Feng T et al (2011)
Polymorphism
of
bone
morphogenetic protein 4 gene and its
relationship with litter size of Jining
Grey goats. Mol Biol Rep (38):
4315 4320.
Dekkers CM (2012) Application of
Genomics
Tools
to
Animal
Breeding. Curr Genomics 13(3):
207 212.
Dhillo, WS, Chaudhri, OB., Patterson, M,
Thompson, EL, Murphy, K. G.,
Badman, M. K.,McGowan, B. M.,
Amber, V., Patel, S., Ghatei, M. A.
and Bloom, S. R. (2005) Kisspeptin54 stimulates the hypo-thalamicpituitary gonadal axis in human
males. J Clin Endocrinol Metab, 90:
6609 6615
Feng T, Geng CX, Lange XZ et al (2011)
Polymorphisms of caprine GDF9
gene and their association with litter
size in Jining Grey goats. Mol Bio
Rep (38): 5189 5197.
Feng T, Chu MX, Cao G L et al (2012)
Polymorphisms of caprine POU1F1
gene and their association with litter
size in Jining Grey goats. Mol Biol
Rep (39): 4029 4038.
Gottsch ML, Cunningham MJ, Smith JT,
Popa SM, Acohido BV, Crowley
WF, Seminara S, Clifton DK and
Steiner RA (2004) A role for
kisspeptins in the regulation of
gonadotropin secretion in the mouse.
Endocrinol 145: 4073-4077.
Huang DW, Wang JX, Liu QY et al. (2013)
Analysis on DNA sequence of TSHB
gene and its association with
reproductive seasonality in goats.
Mol Biol Rep (40)1893 1904.
Huijbregts L, Roze C, Bonafe G, Houang M,
Le Bouc Y, Carel JC, Leger J,
Alberti P, Roux N (2012) DNA
polymorphisms of the KiSS1 3 '
Untranslated region interfere with
the folding of a G-rich sequence into
G-quadruplex.
Mol
Cell
Endocrinol 351:239-248.
Hou JX, An XP, Wang JG, Song YX, Cui
YH, Wang YF, Chen QJ, Cao BY
(2011) New genetic polymorphisms
of KISS-1 gene and their association
with litter size in goats. Small
Ruminant Res 96:106-110.
Hou J, An X, Li J, Wang Y, Song Y, Cao B
(2012) Exploring polymorphisms
and their effects on reproductive
traits of the INHA and INH A genes
801
Int.J.Curr.Microbiol.App.Sci (2015) 4(12): 795-802
in three goat breeds. Ani Sci
(83):73 278.
Ohtaki T, Shintani Y, Honda S, Matsumoto
H, Hori A, Kanehashi K, Terao Y,
Kumano S, Takatsu Y, Masuda Y,
Ishibashi Y, Watanabe T, Asada M,
Yamada T, Suenaga M, Kitada C,
Usuki S, Kurokawa T, Onda H,
Nishimura
O,
Fujino
(2001)
Suppressor gene KiSS-1 encodes
peptide ligand of
a G-proteincoupled receptor. Nature (411): 613617.
Sambrook J, Fritsch EF and Maniatis T
(1989). Molecular Cloning: A
Laboratory Manual. Cold Spring
Harbor Laboratory Press, Nova York
Smith JT, Popa SM, Clifton DK, Hoffman
GE, et al. (2006). Kiss1 neurons in
the forebrain as central processors
for generating the preovulatory
luteinizing hormone surge. J.
Neurosci. 26: 6687-6694.
Tomikawa J, Homma H, Tajima S, Inamoto
Y, Takase K,Inoue N, Ohkura S,
Uenoyama Y, Maeda K, Tsukamura
H (2010) Molecular characterization
and
estrogen
regulation
of
hypothalamic KISS1 gene in the pig.
Biol. Reprod 82:313 319.
Wang PQ, Deng LM, Zhang BY, Chu MX ,
Hou JZ (2011) Polymorphisms of the
cocaine-amphetamine-regulated
transcript (CART) gene and their
association with reproductive traits
in Chinese goats. Genet Mol Res
(10)731 738
Zeshmarani S, Dhara KC, Samanta AK,
Samanta R, Majumder SC (2007)
Reproductive performance of goats
in Eastern and Northeastern India.
Livest. Res. Rural Dev 19: 8
802